骨髓神经干细胞联合Fasudil治疗PD的细胞和分子机制探讨

BM-NSCs, upon entering the CNS, can preferentially migrate into disease foci, where they exert therapeutic effects. However, the effect of this therapy is not yet ideal, most likely due to two reasons: 1) the sustained inflammation and oxidation in the disease areas, making a hostile microenvironment for neural cells; 2) the slow migration of NSCs. Therefore, pharmacologic strategies targeting inflammation process and increasing NSC migration efficiency will significantly enhance therapeutic efficacy of NSCs in CNS disorders. Previous studies have demonstrated that Fasudil, a selective inhibitor of Rho kinase activity, exerts neuroprotective effects, and intranasal (i.n.) administration has recently been found to be a more rapid and effective approach to deliver medications or cells into the CNS than other routes, e.g., oral, i.v. or i.p. .Based on these interesting knowledges, we will in this project explore the efficacy and mechanism of combination of Fasudil with intranasal administartion of BM-NSCs as a novel therapy of MPTP-PD mice. We will study the therapeutic effect by behavioral experiment, dopaminergic neurons surival, α-synuclein accumulation in the substantia nigra (SN) area of brain, endogenous (GFP-) stem cell mobilization, the morphology and function of synapses, neurotransmitter release, and the survival, distribution and differentiation of transplanted (GFP+) BM-NSCs. We then explore the therapeutic mechanism by inflammatory, oxidation stress, neurotrophic factors secret, and apoptosis pathway.. We believe that Fasudil+ nasal delivery of BM-NSCs may be potentially useful therapies that are worth further exploring.

骨髓源神经干细胞（BM-NSCs）可部分抑制中枢神经系统(CNS)神经变性疾病的发展。然而，由于细胞移植的效率低，病理状态下脑内微环境恶劣，干细胞存活率较低，影响其治疗效果。本课题组发现Rho激酶抑制剂盐酸法舒地尔（Fasudil）具有提升干细胞性能，抑制CNS内炎性反应和改善微环境；而且，鼻腔移植途径可使细胞更多更快地进入CNS。为此，拟以腹腔注射Fasudil作为鼻腔移植（GFP+）BM-NSCs的伴侣，探讨Fasudil联合BM-NSCs对MPTP-PD的治疗效果和机制。效果评价指标包括：行为学；病理学；内源性干细胞动员；神经突触形态和功能保护；CNS内外源性（GFP+）干细胞存活分布及分化；神经递质释放；机制探讨从炎症、氧化应激、神经营养因子、凋亡等方面着手。相信BM-NSCs联合Fasudil直接和间接地产生协同或叠加效应，能显现更加理想的治疗效果，有可能成为PD治疗的潜在方案。

中枢神经系统（central nervous system, CNS）变性疾病，在我国发病率逐年提高，目前仍然缺乏有效的治疗方法，骨髓来源的NSCs(bone marrow- derived NSCs，BM-NSCs)，具有和脑源性NSCs相同的特性，BM-NSCs移植技术有望在修复损伤的神经细胞和功能重建中发挥作用，鼻腔途径可以有效提高细胞向CNS定向迁移的效率，Rho激酶抑制剂Fasudil具有优化NSCs的潜能，促进神经保护性微环境的形成。本课题以腹腔注射Fasudil作为鼻腔移植（GFP+）BM-NSCs的伴侣，探讨Fasudil联合BM-NSCs对MPTP-PD的治疗效果和机制。结果显示：Fasudil、NSCs单独治疗组对MPTP-PD显示了明显的治疗效果，Fasudil联合BM-NSCs展示了明显的叠加效应。Fasudil联合BM-NSCs明显改善MPTP-PD小鼠的综合运动能力、缓减了小鼠的焦虑引发的肌肉紧张；更好地保护了黑质多巴胺能神经元分布的完整性；神经保护机制可能与Fasudil可以促进NSCs的增殖、存活，明显减少胶质细胞聚集、抗炎、抗氧化以及调节NMDAR和AMPAR之间的平衡有关。另外，Fasudil联合BM-NSCs也明显地减轻了EAE小鼠的临床症状，减少了髓鞘丢失，抑制了炎性细胞浸润，机制可能与抑制NF-κB炎性信号通路和增加神经营养因子NT-3、GDNF、BDNF、CNTF和NGF的产生有关。因此，我们推断Fasudil联合BM-NSCs对于PD和MS将是一个有效的治疗手段。